The proposed research is the continuation of ongoing studies of the mechanisms of action and regulation of glucose 6-phosphate dehydrogenases (G6PD) from the lactating rat mammary gland and from Leuconostoc mesenteroides. Our projected investigations with the mammary enzyme involve detailed comparisons between G6PD from healthy, lactating rats and G6PD from R3230 AC adenocarcinomas from the same species. Kinetic parameters, extent of inhibition by certain steroids, and polyacrylamide gel electrophoresis band patterns will be compared. Our goal is to provide clearly defined criteria for distinguishing normal from malignant tissue. Both enzymes will be purified to homogeneity and the structural basis for the differences between them will be investigated. Our investigations on the bacterial enzymes will be concerned with isolating the active site peptides, sequencing them and contrasting these sequences with those of other hydrogenases. We will also examine the structural basis for the differences we have found between binding of NAD ion and NADP ion to L. mesenteroides G6PD as well as the differences between the kinetic mechanisms of the NAD- and NADP-linked reactions. Detailed kinetic studies involving coenzyme analogs as well as coenzyme- and substrate-competitive inhibitors will be conducted with the aim of elucidating differential regulatory effects on the reaction with NAD ion and NADP ion. BIBLIOGRAPHIC REFERENCES: Milhausen, M., and H.R. Levy (1975), Evidence for an essential lysine in glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides. Eur. J. Biochem., 50, 453-461. Miller, A.L. and H.R. Levy (1975), Acetyl-CoA carboxylase from rat mammary gland. In: S.P. Colowick and N.O. Kaplan, Methods in Enzymology, 35B, 11-17.